The objective of this proposal is to investigate erythropoietin (EPO) and erythropoietin receptor (EpoR) expression and function in the pathobiology breast cancer. EPO is a hypoxia-inducible glycoprotein hormone that is the principal hematopoietic cytokine regulating the production of red blood cells by binding to its specific cell surface receptor EpoR, a member of the Type I cytokine receptor family. Recombinant human EPO is widely used in patients with cancer for treatment or prevention of anemia associated with chemotherapy and radiation therapy. Recent studies in our laboratory revealed high levels of functional EpoR expression in breast cancer cells. Induction of EpoR expression was observed in human mammary epithelial cells and characterization of the structure of EpoRs revealed the expression of novel EpoR mRNA isoforms in breast cancer. The co-expression of EpoR and its ligand EPO in tumor cells suggested the potential for generation of an autocrine or paracrine growth stimulatory loop. Inhibition of EpoR signaling in vitro or in vivo resulted in decreased proliferation of breast cancer cells suggesting the presence of a previously unknown, functional role for EpoR signaling in the pathobiology of breast cancer. The studies in this proposal will aim to (1) Characterize the role of EPO-EpoR expression and signaling in the proliferation of breast cancer cells and induction of tumor angiogenesis in vivo (2) Investigate the ability of EpoR-mediated signaling to stimulate the proliferation of primary human mammary epithelial cells and (3) Determine whether EPO-EpoR over-expression is coupled to tumor hypoxia and serves as an adverse prognostic factor in patients with breast cancer. We propose to study mechanisms of EPO-EpoR expression and activation in breast cancer cells and investigate the role of EPO-EpoR in tumor growth and angiogenesis using well-established human breast cancer xenografis and rodent mammary windows for orthotopic breast cancer. We will over-express EpoR in primary human mammary epithelial cells (HMECs) to investigate the activation of EpoR signaling pathways in HMECs and the effects on cellular proliferation in an experimental model of early mammary carcinogenesis. We will characterize the role of tumor hypoxia in EPO-EpoR over-expression in primary breast tumors pre-labeled with a cellular hypoxia marker. We shall determine the utility of high levels of EPO-EpoR co-expression in primary tumors as a prognostic factor that may be linked to an unfavorable disease-free and overall survival in patients with breast cancer. It is anticipated that the findings of these studies will contribute to our understanding of the role of EPO and EpoR in the pathobiology of breast cancer and may lead to the formulation of new therapeutic strategies targeted against EpoR function in the future.